If whole-genome sequencing (WGS) is like clicking through a DVD frame by frame, and if fluorescence in situ hybridization (FISH) is like scanning the scene-selection menu, a new technology may offer an intermediate-viewing option. This new technology, high-speed atomic force microscopy (HS-AFM) nanomapping, can create images of up to a million base pairs in size. Although HS-AFM nanomapping cannot match the single-base-pair resolution of DNA sequencing, it has the virtue of greater speed. It can fast-forward to DNA maps offering a resolution of tens of base pairs.

Although HS-AFM nanomapping hasn’t appeared in any high-production-value trailers, a coming attraction of sorts became available November 21 in Nature Communications, in an article entitled, “DNA nanomapping using CRISPR/Cas9 as a programmable nanoparticle.” This article, which was submitted by Virginia Commonwealth University scientists, suggests that HS-AFM nanomapping may fill technical gaps that are poorly addressed by existing DNA-mapping techniques.

The article also explains how HS-AFM nanomapping represents the merger of technical advances in DNA nanotechnology and single-molecule genomics. “We describe a labeling technique (CRISPR/Cas9 nanoparticles) for high-speed AFM-based physical mapping of DNA,” wrote the article’s authors, “and the first successful demonstration of using DVD optics to image DNA molecules with high-speed AFM.”

The Virginia Commonwealth University scientists, led by physicist Jason Reed, Ph.D., anticipate that their new nanomapping technology could transform the way disease-causing genetic mutations are diagnosed and discovered. To demonstrate the nanomapping technology, Dr. Reed and colleagues mapped the genetic translocations present in lymph node biopsies of lymphoma patients.

While there are many potential uses for this technology, Dr. Reed and his team expect that they will, in the immediate future, remain focused on medical applications. They are currently developing software based on existing algorithms that can analyze patterns in sections of DNA up to and over a million base pairs in size. Once the software is completed, it may be combined with a shoe-box-sized instrument to give pathology labs another way to diagnose and evaluate the treatment of diseases linked to genetic mutations.

DNA sequencing is so precise that it can analyze individual base pairs of DNA. But to analyze large sections of the genome to find genetic mutations, technicians must determine millions of tiny sequences and then piece them together with computer software. In contrast, biomedical imaging techniques such as FISH can only analyze DNA at a resolution of several hundred thousand base pairs.

Dr. Reed's new HS-AFM method can map DNA to a resolution of tens of base pairs while creating images up to a million base pairs in size. And it does it using a fraction of the amount of specimen required for DNA sequencing.

“DNA sequencing is a powerful tool, but it is still quite expensive and has several technological and functional limitations that make it difficult to map large areas of the genome efficiently and accurately,” said Dr. Reed, who is a member of the Cancer Molecular Genetics research program at VCU Massey Cancer Center. “Our approach bridges the gap between DNA sequencing and other physical mapping techniques that lack resolution. It can be used as a stand-alone method or it can complement DNA sequencing by reducing complexity and error when piecing together the small bits of genome analyzed during the sequencing process.”

IBM scientists made headlines in 1989 when they developed AFM technology and used a related technique to rearrange molecules at the atomic level to spell out “IBM.” AFM achieves this level of detail by using a microscopic stylus—similar to a needle on a record player—that barely makes contact with the surface of the material being studied. The interaction between the stylus and the molecules creates the image. However, traditional AFM is too slow for medical applications and so it is primarily used by engineers in materials science.

“Our device works in the same fashion as AFM, but we move the sample past the stylus at a much greater velocity and use optical instruments to detect the interaction between the stylus and the molecules,” explained Dr. Reed. “We can achieve the same level of detail as traditional AFM but can process material more than a thousand times faster.”

Because HS-AFM nanomapping uses optical equipment found in DVD players, the technology may be readily mainstreamed. “High-speed AFM is ideally suited for some medical applications as it can process materials quickly and provide hundreds of times more resolution than comparable imaging methods,” noted Dr. Reed.

Increasing the speed of AFM was just one hurdle Dr. Reed and his colleagues had to overcome. To actually identify genetic mutations in DNA, they had to develop a way to place markers or labels on the surface of the DNA molecules so they could recognize patterns and irregularities. An ingenious chemical barcoding solution was developed using a form of CRISPR technology. Reed's team altered the chemical reaction conditions of the CRISPR enzyme so that it only sticks to the DNA and does not actually cut it.

“Because the CRISPR enzyme is a protein that's physically bigger than the DNA molecule, it's perfect for this barcoding application,” stated Dr. Reed. “We were amazed to discover this method is nearly 90 percent efficient at bonding to the DNA molecules. And because it's easy to see the CRISPR proteins, you can spot genetic mutations among the patterns in DNA.”

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